Full-Length cDNA Synthesis Gets an Upgrade

It is well established that one gene codes for more than one protein. As such, it is important to have 5'- and 3'-sequence information in order to learn more about the different transcripts that result from a single gene. Full-length cDNA offers specific information about these end sequences, and thus provides better data about transcriptional variants. Researchers often rely on Rapid Amplification of cDNA Ends (RACE) to acquire full-length sequence of RNA transcripts. If you are working with only a few genes, RACE is more economical than Next-Generation Sequencing (NGS) for this purpose.

To enhance the RACE procedure, Clontech created a kit that incorporated its own SMART (Switching Mechanism at the 5' end of RNA Template) technology. The resulting SMARTer RACE cDNA Amplification Kit has been widely used for both 5'- and 3'-RACE applications, and cited in hundreds of publications. Now, we have developed a new kit—with the same unique SMARTer core, plus further refinements in reaction conditions and for downstream cloning. This new SMARTer RACE 5'/3' Kit outperforms its predecessor in both ease of use and performance (Figures 1 and 2).

Benefits of SMARTer cDNA Synthesis

The new RACE kit retains all the benefits of the previous kit, including the advanced first-strand cDNA synthesis technology, which makes the process fast and relatively simple. The new kit provides higher sensitivity and specificity, as well as lower background (Figure 2), by pairing the SMARTer II A Oligonucleotide with SMARTScribe Reverse Transcriptase. SMARTer first-strand cDNA can be used directly in 5'- and 3'-RACE PCR reactions, without the problematic adaptor ligation step other RACE kits incorporate.

Building on the core SMARTer technology, the new kit’s protocol has been modified to accommodate a larger RNA input volume, and include a larger reaction volume, both of which allow for easier handling.

A Complete Solution for RACE: Synthesize, Clone and Identify with a Single Kit

SMARTer RACE 5'/3' Kits contain all the necessary components (save gene-specific primers) to streamline your process. With this single kit, you can begin first-strand cDNA synthesis and proceed through cloning RACE fragments—all in just 8 hours, enabling recovery of successful clones for analysis on the second day (Figure 1).

Figure 1. Overview of the SMARTer RACE 5'/3' kit workflow. Each kit is a complete system, containing the reagents required to recover cloned RACE fragments on the second day.

In addition to the critical SMARTer reagents, the kit includes SeqAmp DNA Polymerase, which provides robust PCR performance, especially in the amplification of challenging targets that are long or GC-rich (Figure 2).

Figure 2. Comparing products from the new and old SMARTer RACE kits. Primers were designed using the recommendations in each kit’s manual. The samples on this gel image represent a wide range of expression values, and each one has a combined exon length of >10 kb. The new kit is much more successful in amplifying strong, single bands across the sample set. For more data on RACE fragments amplified with the new kit, including target identities, sizes, and GC content, please download the full tech note.

A linearized cloning vector compatible with In-Fusion technology is provided in the kit, and the included primers are designed to specifically match this vector. You must also design your own gene-specific primers according to our listed guidelines to ensure that only your desired 5'- or 3'-RACE fragment is cloned. When used together, the highly efficient natures of SMARTer cDNA synthesis and In-Fusion cloning create an extremely fast, optimized system that takes you from RNA to successful clones, ready for analysis, in just two days.